1. Field of the Invention
The present invention relates generally to the field of protein chemistry. More specifically, the present invention discloses substrates and inhibitors of plague plasminogen activators and their use in detecting Yersinia pestis and controlling the infection caused by Yersinia pestis, respectively.
2. Description of the Related Art
Yersinia pestis, a Gram-negative bacterium is the causative agent of plague, an acute and lethal disease. Although plague is a zoonotic infection, it could be transmitted to humans via a bite from a flea that previously fed on an infected rodent. Typically, flea transmission of Yersinia pestis causes a form of disease referred to as bubonic plague. From the initial site of infection, bacteria disseminate to the draining lymph node, causing swelling of this lymph node to form a bubo, from which, if left untreated, can spread into the circulation, eventually causing bacteremia and the second form of the disease, septicemic plague. Sometimes septicemic disease occurs even without the development of buboes and is characterized by an elevated temperature, chills, headache, malaise and gastrointestinal disturbances.
In addition, pneumonic plague can result if the lungs become infected. Pneumonic plague is the most feared form of the disease that arises due to colonization of the alveolar spaces, and can also be caused by bacterial spread from an infected person (or animal) to a healthy individual by the aerosol route. Pneumonic plague develops rapidly (1-3 days), results in a high mortality rate in infected individuals (approaching 100%), and spreads rapidly from human-to-human. Yersinia pestis is responsible for at least three pandemics in the past, killing by estimation more than 200 million people (Perry et al. 1997). For that reason, and because plague is characterized as an emerging infectious disease, the Centers for Disease Control and prevention has classified it as a category A biological agent. For these reasons, the development of highly effective anti-plague treatments, particularly to combat Yersinia pestis resistant to traditional drugs is an immediate public health priority.
Yersinia pestis contains a unique, 9.5-kb plasmid pPCP that determines four known biochemical functions: a bacteriocin called pesticin, immunity to pesticin and fibrinolytic and coagulase activities. Subsequent studies showed that the latter two activities reside in a single gene encoding an outer membrane protein called plague plasminogen activator (Sodeinde and Goguen., 1988). Plague plasminogen activator expression is associated with the marked ability of Yersinia pestis to colonize the viscera and thus cause lethal infection upon administration by peripheral, i.e. intradermal, subcutaneous or intraperitoneal, routes of infection (Sodeinde et al., 1992).
The importance of plasminogen activator for plague pathogenesis was verified with isogenic plasminogen activator mutants of epidemic Yersinia pestis strains KIM and CO92, which showed up to 106 logs reduced virulence by the subcutaneous route (Sodeinde et al., 1992; Welkos et al., 1997). Since the plague is transmitted to humans via a fleabite, Yersinia pestis exhibits remarkably efficient spreading from the peripheral site of the fleabite to the draining lymph node. This spreading is followed by replication and further invasion of the circulation. The major role in this process has been attributed to plasminogen activator because this plague microbe protease resembles mammalian plasminogen activators in function by converting plasminogen to plasmin by limited proteolysis (Sodeinde and Goguen., 1989), possibly leading to clarification of fibrin deposits that could hinder bacterial migration in circulation (Beesley et al., 1967). Additionally, it was also shown that plasminogen activator can directly inactivate major plasmin inhibitor α2-antiplasmin (α2AP) (Kukkonen et al., 2001) and mediate adhesion to eukaryotic cells (extracellular matrices and basement membranes) which invasive bacteria must penetrate in order to reach the circulation (Lähteenmäki et al., 1998; Lähteenmäki et al., 2001). Finally, recent work using plasminogen-deficient mice has proven importance of plasminogen activation in the pathogenesis of plague, since such mice had a 100-fold increase in the LD50 compared to the normal mice (Goguen et al., 2000). In addition to its role in adhesion, invasion and tissue damage, plasminogen activator has been reported to cleave complement component C3 (Sodeinde et al., 1992), to possess weak coagulase activity (Beesley et al., 1967) and to mediate the proteolysis of yersinia virulence factors (Yops) (Sodeinde et al., 1988). Moreover, a significant antibody response to plasminogen activator was induced after experimental plague infection in mice that survived lethal Yersinia pestis aerosol challenge following antibiotic treatment (Benner et al., 1999); human convalescent sera from plague patients contained antibodies to plasminogen activator as well (Easterbrook et al., 1995).
The plague plasminogen activator of Yersinia pestis is an outer membrane protein, which belongs to the omptin family of bacterial proteases that includes OmpT of E. coli, PgtE of Salmonella and SopA of Shigella flexneri (Lähteenmäki et al., 2001b). However, in contrast to the plague plasminogen activator, the three proteases do not possess the ability either to activate plasminogen or to degrade α2-antiplasmin (Kukkonen et al., 2001), although a recent study suggested that Pgt might have plasminogen-activating capability, which normally stays cryptic for Salmonella (Kukkonen et al., 2004). Further, the predicted structure of plasminogen activator is highly similar to that of its OmpT homolog and has a comparative β-barrel topology with 10 transmembrane β-strands and five surface-exposed loops (Kukkonen et al., 2004). Although plague plasminogen activator is widely referred to as being a serine protease (Lähteenmäki et al., 2001b), the recently resolved structure of homologous OmpT contradicts such a classification (Vandeputte-Rutten et al., 2001). The model predicts that the omptins may constitute a novel class of proteases that is consistent with the observation that commonly used protease inhibitors do not weakly affect the activity of OmpT. Most likely, plague plasminogen activator is not a serine but rather an aspartate protease as predicted for OmpT (Vandeputte-Rutten et al., 2001).
Furthermore, the plasminogen activator protein of Yersinia pestis is significantly different from both mammalian plasminogen activators for example, tissue-type and urokinase, which are both serine proteases that are secreted in a single form and processed proteolytically into a fully active two-chain form (Lähteenmäki et al., 2001b). Crystal structures of the catalytic domains of tissue-type plasminogen activator and urokinase plasminogen activator have been resolved; their overall structures exhibit the typical serine proteinase fold, with insertion loops around the active site cleft determining their specificity for plasminogen. Therefore, the mammalian Pas and plague plasminogen activator of Yersinia pestis represent a classical case of totally unrelated enzymes that show a similar specificity towards the substrate (plasminogen cleavage resulting in a conversion to plasmin). Thus, compounds inhibiting plasminogen activator activity are unlikely to have any effect on the mammalian blood coagulation system.
Thus, features of plasminogen activator such as its surface location, immunogenicity, the existence of a predicted 3D-model and its involvement in Yersinia pestis systemic infection make this protein an excellent candidate to target for development of non-antibiotic therapeutics that are efficacious against plague infection. Despite this, neither substrate specificity nor inhibitors have been found or predicted for the plasminogen activator enzyme.
Thus, prior art is deficient in the knowledge regarding specific substrates and inhibitors of plague plasminogen activator and their use in the detection of Yersinia pestis and in the treatment of Yersinia pestis infection, respectively. The current invention fulfils this long-standing need in the art.